The invention relates to the genetic manipulation of plants, particularly to altering metabolic end products in plants and plant seeds.
A long time goal of scientists has been to improve the fatty acid profile for oils. The oxidative stability of the vegetable oil is related to the number of double bonds in its fatty acids. That is, molecules with several double bonds are recognized to be more unstable. Thus, scientists have attempted to reduce the amount of alpha-linolenic acid in order to improve shelf life and oxidative stability. Unfortunately, the use of naturally occurring germplasm has not proven to be successful by traditional breeding mechanisms.
Other work has concentrated on producing modified oils with specific fatty acid composition, particularly very high oleic acid oils. High-oleic canola, safflower, and sunflower oils as well as low-linolenic canola and soybean oils are on the market, although in limited quantities.
Modified oils are needed because of the stability as well as for health concerns. For example, high-oleic sunflower oil has about ⅓ the saturated fat content of cottonseed oil.
A major source of fatty acids is biosynthesis from small-molecule intermediates derived from metabolic breakdown of sugars, some amino acids, and other fatty acids. Acetyl-CoA is the direct source of all carbon atoms for the synthesis of palmitic acid. In a majority of instances the saturated straight-chain C16 acid, palmitic acid, is first synthesized and all other fatty acids are made by modification of palmitic acid. Fatty acids are synthesized by sequential addition of 2-carbon units to the activated carboxyl end of a growing chain.
Acetyl-CoA Carboxylase (ACCase) catalyzes the formation of malonyl-CoA from acetyl-CoA and bicarbonate in animal, plant, and bacterial cells. Malonyl-CoA is an essential substrate for (i) de novo fatty acid synthesis, (ii) fatty acid elongation, (iii) synthesis of secondary metabolites such as flavonoids and anthocyanins, and (iv) malonylation of some amino acids and secondary metabolites. Synthesis of malonyl-CoA is the first committed step of flavonoid and fatty acid synthesis and current evidence suggests that ACCase catalyzes the primary regulatory or rate-limiting step of fatty acid synthesis. Formation of malonyl-CoA by ACCase occurs via two partial reactions and requires a biotin prosthetic group:
E-biotin+ATP+HCO3xe2x86x92E-biotin-CO2+ADP+Pi
(ii) E-biotin-CO2+Acetyl-CoAxe2x86x92E-biotin+malonyl-CoA
(NET) Acetyl-CoA+ATP+HCO3xe2x86x92malonyl-CoA+ADP+Pi
In bacteria such as Escherichia coli, the ACCase has four distinct, separable protein subunit components; a biotin carboxyl carrier protein, a biotin carboxylase and two subunits of carboxyltransferase. In eukaryotes, ACCase is composed of multimers of a single multifunctional polypeptide having a molecular mass typically greater than 200 kDA (Samols et al., J. Biol. Chem. 263:6461-6464 (1988)). These multimers have molecular masses ranging from 400 kDa to 8 MDa.
De novo fatty acid synthesis in chloroplasts involves successive 2-carbon additions to acetate, using malonate as the 2-C donor. All intermediates are attached to acyl carrier protein (ACP). Synthesis in plastids resembles that in E. coli in that the fatty acid synthesis complex can be dissociated into separate enzymes: xcex2-ketoacyl-ACP synthase (KAS), xcex2-ketoacyl-ACP reductase, xcex2-hydroxyl-ACP dehydratase, and enoyl-ACP reductase, acetyl-CoA:ACP transacylase, and malonyl-CoA:ACP transacylase. A highly active KASIII isozyme catalyzes the condensation of acetyl-CoA and malonyl-ACP. Successive additions of malonyl-CoA to acyl-ACPs catalyzed by KASI form C16 acyl-ACP, some of which is converted to C18 acyl-ACP by KASII and then to C18:1-ACP. Fatty acid metabolism then diverges. De-esterification allows movement to the cytoplasm (eukaryotic path) where fatty acids may be further unsaturated and/or elongated by additions of malonyl-CoA in the ER. Alternatively, fatty acids are linked to glycerol-3 phosphate (prokaryotic path), further unsaturated, and used for synthesis of chloroplast lipids. A portion of cytoplasmic lipids returns to the chloroplast. The relative contributions of these two paths are species-specific but appear to be relatively flexible in mutants blocked in either path. In oil-storing organs such as cotyledons and monocot embryos, the triacylglycerides are stored in cytoplasmic oil bodies surrounded by a single unit membrane.
Lipids, particularly triglycerides, have a great deal of commercial value in food and industrial products. Sunflower, safflower, rape, olive, soybean, peanut, flax, castor, oil palm, coconut and cotton are examples of major crops which are grown primarily or secondarily for their lipids. All agricultural animals provide animal sources for commercial fats and oils.
Seeds contain oil, starch, and protein in proportions which depend upon the plant species, cultivar and the stage of development. In the mature seed of oil seed rape, the main storage products are oil and protein, but starch accumulates transiently during the early phase of oil deposition. In non-photosynthetic tissues the synthesis of starch and fatty acids occurs in the plastids and requires the supply of carbon precursors from the cytosol.
There is needed a method for producing significant levels of fatty acids, carotenoids and other metabolic products in plants, particularly plant seeds. Such altered seeds would be useful nutritionally as well as provide a source for specialty oils and compounds.
Compositions and methods for modulating a metabolic pathway of a cell, particularly those pathways utilizing acetyl-CoA as a starting material, are provided. Such metabolic pathways include fatty acid biosynthesis, synthesis of isoprenoid compounds, and production of amino acids. The compositions comprise nucleotide sequences encoding the enzyme acetyl-CoA synthetase (ACS). ACS catalyzes formation of acetyl-CoA. Acetyl-CoA is a precursor to fatty acids in plastids through a four-step process using the substrate acetate.
The compositions and methods find use in increasing biosynthesis of fatty acids and/or carotenoids in plants. Thus, utilizing the methods of the invention, seed can be obtained having increased levels of oils, specialty oils, carotenoids, and amino acids genetically altering the content of seed. The methods involve preparing expression constructs with the nucleotide sequences of the invention and the utilization of such constructs to prepare plants having altered metabolic pathways.
The nucleotide sequences of the invention can be used in combination with other genes and antisense sequences to alter the content of a plant seed. In this manner plant seeds having high oil content, specialty oils, increased essential amino acids, carotenoids and other metabolites can be obtained.